Project Summary Diagnosed mainly based on symptomatic description, it is difficult to distinguish the underlying mechanisms of neuropsychiatric disorders, including schizophrenia (SCZ) and autism spectrum disorders (ASD). Recent advanced human genetic studies have identified the genetic underpinning of the a variety of neuropsychiatric disorders, showing that majority of SCZ and ASD are genetically heterogeneous and caused by with rare de novo mutations. Particularly, a rare mutations at SET1/COMPASS complex proteins or related proteins were distinctly discovered at SCZ or ASD patients. Our central hypothesis is that investigating the function of SCZ- or ASD-associated proteins in cortical and subcortical development will reveal the molecular mechanism how mutations of proteins with similar catalytic activity result in clinically distinct disorders. The paucity of accessible human brain tissues has been challenging to directly addressing these questions by using human brain tissue. Thus, in order to achieve the goal, we will use human brain organoids that structually and functionally reproduce the developing human brain regions. The use of human pluripotent stem cells (hPSCs) has revolutionized the human brain studies. hPSCs undergo unlimited self-renewal and can differentiate into any cell types, including brain cells. We have reported the generation of 3-dimensional (3-D) structures from hPSCs that recapitulate the developing human cortex (hCO, cortical organoids), medial ganglionic eminence (hMGEO, MGE organoid), or diencephalic thalamus (hThO, thalamic organoids). Fusing hCO with hMGEO, or hThO reproduced the interaction of developmentally distinct two regions, such as tangential migration of MGE cells to cortex, or reciprocal corticothalamic or thalamocortical connections. Using the advanced stem cell tools, we will purse the aims to achieve the goal. 1) We will use CRISPR/CAS9 gene editing tools to introduce mutations of SCZ and ASD genes into hPSC lines, and investigate the cellular and molecular function in cortical and thalamic development using hThOs and hCOs. 2) We will develop methods to reproduce the multiple nuclei in thalamus to further improve the regional specification of hThOs. 3) We will investigate the function of SCZ and ASD genes in cortical and subcortical connectivity. Overall, our advanced human brain organoid-based approaches combined with genomics and neurobiological tools will define the molecular mechanism distinctly regulated by SCZ and ASD-associated genes and provide unprecedented unique platforms to construct the functional corticothalamic connection.